Mode of action of RNA/DNA oligonucleotides: progress in the development of gene repair as a therapy for alpha(1)-antitrypsin deficiency.

We describe a technology developed for the site-specific correction of a single base carried on an episome or chromosome in prokaryotic and eukaryotic cells. Critical to the development of this technology as a therapeutic device for treating genetic disorders, like alpha(1)-antitrypsin deficiency, is the establishment of a standardized assay to study its mode of action and structure-activity relationships (SARs). To this end, a positive-selection system in Escherichia coli has been developed to assess RNA/DNA oligonucleotide (RDO)-directed repair activity. We demonstrate that RDO-directed repair requires the concerted action of the two following repair proteins: the pairing protein RecA; and the mismatch recognition protein, MutS. SAR studies demonstrate that the RDO molecule is functionally asymmetric. The RNA-containing strand enables strand-pairing and stabilization of the molecule, and the DNA-containing strand confers the information transfer.